Bacterial pathogens exploit various molecular mechanisms to survive unpredictable and adverse environmental conditions. Gram-negative bacteria often alter their environmentally exposed outer membrane, an asymmetric bilayer consisting of inner leaflet glycerophospholipids and essential outer leaflet lipooligosaccharide or lipopolysaccharide. Acinetobacter baumannii is a Gram-negative nosocomial pathogen that thrives in healthcare settings because of its ability to develop resistance to antibiotics. Multidrg resistant A. baumannii have become widespread over the past decade and last-line antibiotics such as colistin, which target the essential lipooligosaccharide in the outer membrane, have been increasingly prescribed to treat multidrug resistant infections. While colistin resistance was once rare, this is no longer the case, especially regarding A. baumannii. Uniquely, A. baumannii can completely shut down lipopolysaccharide biosynthesis to develop resistance to colistin and many other commonly prescribed antibiotics. This finding is surprising because lipooligosaccharide and lipopolysaccharide were previously thought to be required for Gram-negative bacterial viability, but this multidrug resistance mechanism proves that it is not essential. Mechanisms that contribute to this multidrug resistance phenotype are not understood and treatment options have not been explored. The overall objective of this proposal is to characterize and understand a novel multidrug resistance mechanism. The Specific Aims of this proposal are (i) to understand the genetic requirements for complete loss of lipooligosaccharide and (ii) to characterize the altered outer membrane permeability barrier after complete loss of lipooligosaccharide. Completion of this work will contribute an essential body of knowledge to the essentiality of lipooligosaccharide or lipopolysaccharide in Gram-negative bacteria and provide understanding of a molecular mechanism required for a novel multidrug resistance mechanism. The basic science framework from this proposal could also potentially lead to development of novel therapeutics and improved vaccines.